Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
The isolation of a phenotypically unique subpopulation of cells from a heterogenous cell population based on differential expression of cell surface markers is an essential step in many medical and biological research investigations. In addition, several protocols for the treatment of hereditary or acquired diseases that depend on the efficient selection of subpopulations of cells either are currently being employed or are in clinical trials. For example, in bone marrow (BM) or peripheral blood stem cell transplantation, hematopoietic stem/progenitor cells (HPC) are being purified from harvested BM or leukophoresis products to eliminate contaminating tumor cells or potentially alloreactive T cells (reviewed in Gee 1994; Berenson et al. 1996). In addition, stem cell gene therapy protocols are under development that also rely on HPC selection (Kohn 1995). Thus, it is clear that cell separation technologies are important in basic biological and medical research and in clinical medicine and the importance of this technology is likely to grow in the future.
At present, the available cell separation media employ monoclonal antibodies (Mab) that bind to specific cell surface antigens and are linked to solid (Lebkowski et al. 1992; Bensinger et al. 1990) or magnetic (Egeland et al. 1993; Miltenyi et al. 1990) matrices through covalent means or via biotin/avidin or immunoglobulin/anti-immunoglobulin interactions. The cells are either used without detachment from the matrix or are released by mechanical agitation, enzymatic digestion or dissociation induced by binding of an anti-mouse Fab antibody (Shpall et al. 1994). The mechanical agitation and enzymatic methods for releasing bound cells can result in damage to the targeted cell population and are difficult to monitor. Accordingly, a cell separation methodology is still needed that can separate target cells with specificity and without damaging the target cells.